Biomedical subjects

J Wellink

Publications and source records attributed to J Wellink.

At least 37 records · Page 2Linked to original sources

PubMed1996

Distinct functional domains in the cowpea mosaic virus movement protein.

Cell-to-cell movement of cowpea mosaic virus particles in plants takes place with the help of tubules that penetrate presumably modified plasmodesmata. These tubules, which are built up by the virus-encoded 48-kDa movement protein (MP), are also formed on single protoplast cells. To determine whether the MP contains different functional domains, the effect of mutations in its coding region was studied. Mutations between amino acids 1 and 313 led to complete abolishment of the tubule-forming capacity, while a deletion in the C-terminal region resulted in tubules that could not take up virus particles. From these observations, it is concluded that the MP contains at least two distinct domains, one that is involved in tubule formation and that spans amino acids 1 and 313 and a second that is probably involved in the incorporation of virus particles in the tubule and that is located in the C terminus between amino acids 314 and 331.

Comovirus↗

PubMed1995

The cowpea mosaic virus RNA 1-encoded 112 kDa protein may function as a VPg precursor in vivo.

Processing of the 112 kDa ('112K') protein encoded by cowpea mosaic virus RNA 1 was examined in cowpea mesophyll protoplasts using a transient expression system. Cleavage of the 112K protein occurred via two alternative pathways either into VPg and 110K (24K + 87K) or into 26K (VPg + 24K) and 87K proteins. The 26K protein can be further cleaved into VPg and 24K proteins. The results support a model in which the 112K protein functions as the precursor of VPg during initiation of replication.

Comovirus↗

PubMed1994

Adaptation of positive-strand RNA viruses to plants.

The vast majority of positive-strand RNA viruses (more than 500 species) are adapted to infection of plant hosts. Genome sequence comparisons of these plant RNA viruses have revealed that most of them are genetically related to animal cell-infecting counterparts; this led to the concept of "superfamilies". Comparison of genetic maps of representative plant and animal viruses belonging to the same superfamily (e.g. cowpea mosaic virus [CPMV] versus picornaviruses and tobacco mosaic virus versus alphaviruses) have revealed genes in the plant viral genomes that appear to be essential adaptations needed for successful invasion and spread through their plant hosts. The best studied example represents the "movement protein" gene that is actively involved in cell-to-cell spread of plant viruses, thereby playing a key role in virulence and pathogenesis. In this paper the host adaptations of a number of plant viruses will be discussed, with special emphasis on the cell-to-cell movement mechanism of comovirus CPMV.

Adaptation, Physiological↗

PubMed1994

Replication and translation of cowpea mosaic virus RNAs are tightly linked.

The genome of cowpea mosaic virus (CPMV) is divided among two positive strand RNA molecules. B-RNA is able to replicate independently from M-RNA in cowpea protoplasts. Replication of mutant B-transcripts could not be supported by co-inoculated wild-type B-RNA, indicating that B-RNA cannot be efficiently replicated in trans. Hence replication of a B-RNA molecule is tightly linked to its translation and/or at least one of the replicative proteins functions in cis only. Remarkably also for efficient replication of M-RNA one of its translation products was found to be required in cis. This 58K protein possibly helps in directing the B-RNA-encoded replication complex to the M-RNA. In order to identify the viral polymerase the CPMV B-RNA-specific proteins have been produced individually in cowpea protoplasts using CaMV 35S promoter based expression vectors. Only protoplasts transfected with a vector containing the 200K coding sequence were able to support replication of co-transfected M-RNA. Despite this, CPMV-specific RNA polymerase activity could not be detected in extracts of these protoplasts using a poly(A)/oligo(U) assay. These results indicate that, in contrast to the poliovirus polymerase, the CPMV polymerase is not able to accept oligo(U) as a primer and in addition support the concept that translation and replication are linked.

Animals↗

PubMed1994

The NTP-binding motif in cowpea mosaic virus B polyprotein is essential for viral replication.

We have assessed the functional importance of the NTP-binding motif (NTBM) in the cowpea mosaic virus (CPMV) B-RNA-encoded 58K domain by changing two conserved amino acids within the consensus A and B sites (GKSRTGK500S and MDD545, respectively). Both Lys-500 to Thr and Asp-545 to Pro substitutions are lethal as mutant B-RNAs were no longer replicated in cowpea protoplasts. Transiently produced mutant proteins were not able to support trans-replication of CPMV M-RNA in cowpea protoplasts in contrast to transiently produced wild-type B proteins. Therefore loss of viral RNA synthesis was a result of a protein defect rather than an RNA template defect. Mutant B polyproteins were correctly processed in vitro and in vivo and the regulatory function of the 32K protein on processing of B proteins was not affected by these mutations. Since regulation of processing by the 32K protein depends on interaction with the 58K domain, the mutations in the NTBM apparently do not interfere with this interaction. The Asp-545 to Pro substitution left intact the binding properties of the 84K precursor of the 58K protein, with respect to ATP-agarose, whereas the Lys-500 to Thr substitution decreased the binding capacity of the 84K protein, suggesting that the Lys-500 residue is directly involved in ATP binding. The Lys-500 to Thr substitution in the 58K domain resulted in an altered distribution of viral proteins, which failed to aggregate into large cytopathic structures as observed in protoplasts infected with wild-type B-RNA. However viral proteins containing the Asp-545 to Pro substitution showed a normal distribution in protoplasts.

Adenosine Triphosphate↗

PubMed1993

Cis- and trans-acting elements in cowpea mosaic virus RNA replication.

Cowpea mosaic virus (CPMV) B-RNA encodes the viral proteins required for viral RNA replication while M-RNA does so for the capsid proteins and functions required in cell-to-cell movement of the virus. Accordingly, B-RNA can replicate by itself, whereas M-RNA can only replicate in the presence of B-RNA. We have made heterologous sequence insertions at different positions in the open reading frame of B-RNA, leaving the 5' and 3' non-coding ends intact. None of these mutant B-RNAs were able to replicate. Furthermore, it was not possible to support replication of these mutant B-RNAs by co-inoculating wild-type B-RNA as a helper, indicating that B-RNA can not be replicated in trans. In contrast, replication of M-RNA must occur in trans, as the viral replicative proteins are encoded by B-RNA. Mutant M-RNA transcripts containing 5' and 3' non-coding regions of B-RNA are still efficiently replicated in protoplasts if co-inoculated with B-RNA, indicating that in cis or in trans replication of the CPMV RNAs is not primarily determined by the non-coding regions. Remarkably, for replication of M-RNA, the N-terminal domain of the 58K protein encoded by M-RNA was found to be required.

Base Sequence↗

PubMed1993

Mutational analysis of AUG codons of cowpea mosaic virus M RNA.

The involvement of the AUG codons at positions 115, 161, 512 and 524 in translation and infectivity of cowpea mosaic virus M RNA was studied. Mutations were introduced in each of these codons in a full length cDNA clone of M RNA and the effect of the mutations was examined by translation from in vitro transcripts of these mutant cDNAs in rabbit reticulocyte lysates and by checking the replication of these transcripts in the presence of B RNA in cowpea protoplasts and plants. It was found that AUG115, at the beginning of an open reading frame (ORF) for a putative 2-kDa protein, can be used in vitro to initiate translation, but mutation of this AUG codon in the M RNA does not affect the ability of the virus to infect cowpea plants. AUG161, located at the beginning of the large ORF, was shown to be essential for expression of the large 105-kDa polyprotein and for replication of M RNA. Translation of the second 95-kDa polyprotein was found to start at AUG512. Upon mutation of this AUG codon efficient initiation of translation occurred at AUG524. Results with M RNAs that lack AUG512 and/or 524 indicate that an intact 95-kDa polyprotein is not required for replication of M RNA but that this protein has an essential function in cell-to-cell movement of the virus.

Amino Acid Sequence↗

PubMed1993

Protoplasts transiently expressing the 200K coding sequence of cowpea mosaic virus B-RNA support replication of M-RNA.

In order to identify the viral polymerase involved in cowpea mosaic virus (CPMV) RNA replication the 87K, 110K and 170K proteins as well as the complete 200K polyprotein of CPMV B-RNA have been produced in cowpea protoplasts, using expression vectors based on the 35S promoter of cauliflower mosaic virus. CPMV-specific proteins were obtained that were indistinguishable from proteins found in CPMV-infected protoplasts. Proteolytic processing of precursor proteins synthesized from the expression vectors proved that the 24K protease contained within these proteins is active. Moreover, it was established that protoplasts transfected with the expression vector containing the entire 200K coding sequence, but not those transfected with vectors containing the 170K, 110K or 87K coding sequences, were able to support replication of co-inoculated M-RNA. Despite the ability to support replication of M-RNA for protoplasts transiently expressing the 200K coding region, CPMV-specific RNA polymerase activity dependent on exogenous added template RNA could not be detected in extracts of these protoplasts in assays using poly(A).oligo(U) or other template/primer combinations. In contrast, extracts of protoplasts in which poliovirus polymerase was produced exhibited RNA polymerase activity in such assays. These results indicate that the CPMV polymerase, unlike the poliovirus polymerase, is not able to use oligo(U) as a primer or cannot function on exogenous template and primer RNA.

Base Sequence↗

PubMed1993

The involvement of cowpea mosaic virus M RNA-encoded proteins in tubule formation.

On the surface of cowpea protoplasts inoculated with cowpea mosaic virus (CPMV), tubular structures containing virus particles have been found. Such tubular structures are thought to be involved in cell-to-cell movement of CPMV in cowpea plants. To study the involvement of the 58K/48K and capsid proteins of CPMV in the formation of the tubular structures, mutations were introduced into M cDNA clones from which infectious transcripts could be derived. No tubules were found on protoplasts inoculated with a mutant that fails to produce the 48K protein nor with a mutant that has a deletion in the 48K coding region, suggesting that the 48K protein is essential for this process. However, a possible role of the 58K protein in tubule formation could not be excluded. A mutant that fails to produce the capsid proteins did produce tubules and therefore the capsid proteins are not involved in the formation of the tubular structures. Electron microscopic analysis revealed that the tubules produced by this mutant are, apart from the absence of virus particles, morphologically identical to the tubules formed by the wild-type virus.

Base Sequence↗

PubMed1993

Defective cell-to-cell movement of cowpea mosaic virus mutant N123 is efficiently complemented by sunn-hemp mosaic virus.

During an infection with cowpea mosaic virus (CPMV) both virion assembly and formation of tubules associated with plasmodesmata are required for cell-to-cell movement. These functions are encoded by the M-RNA of CPMV. To study the mechanism of CPMV movement, mutant N123 was used in complementation studies with sunn-hemp mosaic virus (SHMV), a legume-infecting tobamovirus. Previous studies have shown that N123 fails to spread in cowpea plants because of mutation(s) in its M-RNA. However, the mutant was efficiently replicated in cowpea protoplasts, in which virions were formed and tubular transport structures were induced. After high-dose inoculation of cowpeas with N123, only a few infected protoplasts could be isolated, indicating that cell-to-cell transport of N123 was greatly impaired, if not completely abolished. Upon coinoculation with SHMV, mutant N123 infected cowpea plants systemically and accumulated to levels which were comparable to those of wild-type CPMV. In contrast, separate B-RNA of CPMV and a CPMV deletion mutant lacking the tubule-inducing function, were complemented by SHMV to only low levels. It is concluded that SHMV-facilitated spread of CPMV in the non-virion tobamovirus mode is inefficient and that spread of mutant N123 is probably in the CPMV mode, SHMV providing an as yet unidentified helper function.

Brassica↗

PubMed1993

The cowpea mosaic virus M RNA-encoded 48-kilodalton protein is responsible for induction of tubular structures in protoplasts.

Tubular structures extending from plasmodesmata in cowpea mosaic virus (CPMV)-infected tissue have been implicated to play an important role in cell-to-cell movement of this virus. Using a cauliflower mosaic virus 35S promoter-based transient expression vector, we show that expression of only the CPMV M RNA-encoded 48-kDa protein (48K protein) in cowpea protoplasts is sufficient to induce these structures. Strikingly, expression of the 48K protein in protoplasts from a number of nonhost plant species, such as barley, Arabidopsis thaliana, and carrot, also resulted in tubular structure formation. Thus, it is not likely that the viral 48K protein, though playing a key role in cell-to-cell movement of CPMV, has a role in determining the host range of CPMV.

Fabaceae↗

PubMed1992

A regulatory role for the 32K protein in proteolytic processing of cowpea mosaic virus polyproteins.

We have studied the regulation of proteolytic processing of the polyproteins encoded by cowpea mosaic virus M-RNA and B-RNA. For that purpose mutations were introduced in full-length cDNA clones of these RNAs. RNA transcripts were translated in rabbit reticulocyte lysate and the effect of mutations on the processing was analysed. These studies revealed that the 32K protein is released from the 200K B-polyprotein by an intramolecular cleavage and remains associated with the 170K protein, probably by interaction with the 58K domain of the 170K protein. In this complex the conformation of the 170K protein is such that further cleavages are very slow. This complex carries out the processing of the Gln/Met site in the M-polyprotein. The 170K protein produced by a B-RNA mutant that lacks the 32K coding region was efficiently processed into 110K, 87K, 84K, 60K, 58K and 24K cleavage products. Thus, the 32K protein regulates the B-polyprotein processing by slowing it down and, on the other hand, enhances trans cleavage of M-polyproteins at a Gln/Met site.

Base Sequence↗

PubMed1992

Processing of VPg-containing polyproteins encoded by the B-RNA from cowpea mosaic virus.

To study the processing of putative VPg precursors the expression of specific mutant transcripts derived from a full-length cDNA clone of cowpea mosaic virus (CPMV) B-RNA was examined in a rabbit reticulocyte lysate system. This study revealed that the 170K protein produced by a B-RNA mutant that lacks the 32K coding region was efficiently processed by mainly intramolecular cleavages at three different sites into three sets of proteins of 60K + 110K, 84K + 87K, and 58K + 112K. Further cleavage of the 60K protein into 58K and VPg has not been observed in this in vitro system. The 84K protein can be further processed by an intramolecular cleavage reaction via two alternative pathways, either into 26K (VPg + 24K) and 58K proteins or into 24K and 60K proteins. VPg can be released from the 112K (VPg + 110K) precursor either directly or via the 26K intermediate. Immunoblot analysis showed that the 112K protein is present in CPMV-infected plant cells indicating that the in vitro observations may hold true in vivo.

Base Sequence↗

PubMed1992

Synthesis of the complete 200K polyprotein encoded by cowpea mosaic virus B-RNA in insect cells.

The coding sequence for the entire 200K polyprotein of cowpea mosaic virus (CPMV) B-RNA was expressed in insect cells by using baculovirus expression vectors. The 200K polyprotein, which harbours all virus functions required for RNA replication, is completely cleaved into 170K and 32K products by the 24K protease activity contained within the polyprotein. Further processing of the 170K protein into CPMV-specific products of 60K, 84K, 87K, 110K and 112K occurred to a limited extent, similar to that observed in cowpea cells. Electron microscopy of insect cells in which the 200K protein was produced revealed the presence of membranous vesicles and electron-dense structures which were not seen in cells infected with wild-type baculovirus. Similar cytopathic structures develop in the cytoplasm of CPMV-infected cowpea cells and are thought to be the site of membrane-bound viral RNA replication. The electron-dense structures in insect cells could be preferentially labelled with several CPMV-specific antisera and Protein A-gold. Since electron-dense structures were not observed in cells in which the 170K protein only was produced, it seems that the 32K protein has a role in keeping the B-RNA-encoded proteins in these structures together. Membranous vesicles were also observed in insect cells in which the 60K protein only was produced. Use of specific antibodies and Protein A-gold showed that the 60K protein is associated with these vesicles, indicating that the 60K protein may induce the formation of vesicles. Although proteolytic processing of the 200K polyprotein and the induction of cytopathic structures indicate that the CPMV proteins produced in insect cells are functional, it has not been possible to demonstrate RNA polymerase activity in extracts of these cells using an oligo(U)-primed assay. The results indicate that in the assay an additional component is lacking and/or that the CPMV polymerase is not able to start RNA synthesis on an exogenous template.

Animals↗

PubMed1991

The sequence between nucleotides 161 and 512 of cowpea mosaic virus M RNA is able to support internal initiation of translation in vitro.

Cowpea mosaic virus M RNA is translated in vitro as well as in vivo into two C-coterminal polyproteins of Mr 105K and 95K. Initiation of translation of the 95K protein gene occurs at an AUG codon at position 512 of M RNA, 351 nucleotides downstream of the initiation codon of the 105K protein gene at position 161. By employing an in vitro transcription and translation system it was determined that this 351 nucleotide sequence has the capacity to direct ribosomes to initiate translation at a downstream start codon. This effect is independent of the position of this sequence in an mRNA. Furthermore, evidence has been obtained that scanning ribosomes can bypass the AUG at position 161. Thus, both leaky scanning and internal entry are mechanisms for the initiation of translation of the 95K protein gene.

Base Sequence↗

PubMed1991

Tubular structures involved in movement of cowpea mosaic virus are also formed in infected cowpea protoplasts.

In cowpea plant cells infected with cowpea mosaic virus, tubular structures containing virus particles are formed in the plasmodesmata between adjacent cells; these structures are supposedly involved in cell-to-cell spread of the virus. Here we show that similar tubular structures are also formed in cowpea protoplasts, from which the cell wall and plasmodesmata are absent. Between 12 and 21 h post-inoculation, tubule formation starts in the periphery of the protoplast at the level of the plasma membrane. Upon assembly, the virus-containing tubule is enveloped by the plasma membrane and extends into the culture medium. This suggests that the tubule has functional polarity and makes it likely that a tubule 'grows' into a neighbouring cell in vivo. On average, 75% of infected protoplasts were shown to possess tubular structures extending from their surface. The tubule wall was 3 to 4 nm thick and they were up to 20 microns in length, as shown by fluorescent light microscopy and negative staining electron microscopy. By analogy to infected plant cells, both the viral 58K/48K movement and capsid proteins were located in these tubules, as determined by immunofluorescent staining and immunogold labelling using specific antisera against these proteins. These results demonstrate that the formation of tubules is not necessarily dependent on the presence of plasmodesmata or the cell wall, and that they are composed, at least in part, of virus-encoded components.

Capsid↗

PubMed1991

Evidence for dissimilar properties of comoviral and picornaviral RNA polymerases.

The poliovirus RNA polymerase has been synthesized in Spodoptera frugiperda cells by using the baculovirus expression system. Crude sonicates of these cells exhibited an RNA-elongating activity of a synthetic oligo(U) primer with poly(A) or cowpea mosaic virus (CPMV) RNA as a template. A similar polymerase activity was found in extracts of insect cells in which foot-and-mouth disease virus (FMDV) proteins, including the putative polymerase, were produced. The analogous CPMV 87K protein and several of its precursors, synthesized in S. frugiperda cells, did not show any detectable polymerase activity in the same assay under a variety of conditions. The results indicate that, in contrast to the picornaviral polymerases, the CPMV polymerase is unable to function in an oligo(U)-primed polymerase assay.

Animals↗

PubMed1991

Cowpea mosaic virus middle component RNA contains a sequence that allows internal binding of ribosomes and that requires eukaryotic initiation factor 4F for optimal translation.

Cowpea mosaic virus (CPMV) middle component RNA (M-RNA) encodes two proteins of 105 and 95 kDa, of which translation starts at nucleotide (nt) 161 and nt 512, respectively. In vitro translation of both proteins directed by T7 transcripts of M-RNA was stimulated fourfold by eukaryotic initiation factor 4F (eIF-4F), the cap-binding protein complex. The ratio of the synthesis of both proteins after translation was not influenced by eIF-4F or by any known eIF. Part of the CPMV 5' sequence was cloned downstream of the 5' untranslated region of ornithine decarboxylase (ODC); the latter untranslated sequence has a highly stable secondary structure, preventing efficient translation of ODC. Insertion of nt 161 to 512 of CPMV M-RNA upstream of the ODC initiation codon resulted in a marked increase in ODC translation, which indicates that the CPMV sequence contains an internal ribosome-binding site. The insertion conferred stimulation by eIF-4F on ODC translation, showing that eIF-4F is able to stimulate internal initiation.

Base Sequence↗